This research will explore the molecular interactions of phosphoenolpyruvate carboxylase with its regulatory ligands. Experimental emphasis will be focused on the enzyme from Crassula argentea, with comparative studies involving phosphoenolpyruvate carboxylase from E. coli and Z. mays. The metabolic inhibitor malate and the activator glucose 6-phosphate are the principal effectors to be studied. The specific aims include determining which ionization states are important for these effectors, the role of metal ions in effector binding, and what substructures of the effectors are essential for their regulatory activity. Interactions between malate and glucose 6-phosphate will be evaluated. Kinetic evidence of mutually exclusive binding of various inhibitors will be used to address the possibility of multiple inhibitor sites. Evidence will be obtained from chemical modification studies on the types of amino acid residues involved in effector binding, whether the effectors are binding at true allosteric sites, and how many distinct types of effector sites are present. Affinity labels will be developed which should prove to be useful probes of the regulatory sites of this enzymes, as well as bacterial and mammalian enzymes with binding sites for either of these two metabolic intermediates.